Diabetes mellitus (DM) is a major risk factor for the development of cardiovascular disease. The myocardium is a target for hyperglycemia induced oxidative stress, which leads to myocyte cell death by apoptosis and left ventricular (LV) dysfunction. An emerging area of cell biology is the application of gene based strategies to inhibit programmed cell death. The global objective of this approach is to preserve or restore cell number and to prevent or attenuate remodeling. Exciting new data from our laboratory demonstrated PKC epsilon dependent cardioprotection against the hyperglycemia apoptosis signal. An important question concerns the translation of in vitro cytoprotection assays to the in vivo condition of the diabetic myocardium. Specific Aims 1 and 2 will explore this question in the streptozotocin (STZ) DM model, in 2 strains of genetically engineered mice, with cardiac specific expression of PKC epsilon activator (pseudo epsilon-RACK) or a PKC epsilon inhibitor (eV1). Immunocytochemical, biochemical and physiological approaches will be performed to identify the molecular components of the survival program and which genes are essential for PKC epsilon dependent cardioprotection. Under Specific Aim 3, the novel strategy of short interfering RNA (siRNA) will be employed to induce stable loss of function genotypes in primary cultures of adult rat ventricular myocytes (ARVM). The objective will be to identify which of the PKC epsilon signaling molecules are essential for the expression of the survival phenotype. These studies will complement those outlined under Specific Aims 1and 2, as selective peptide activators and inhibitors of PKC epsilon will be delivered to ARVM-null cells maintained under hyperglycemic conditions. We will also explore the role of hyperglycemia induced oxidativestress in the modulation of the PKC epsilon survival signal. Antisense inhibition of the antioxidant protein, thioredoxin in ARVM, followed by delivery of PKC epsilon activators or inhibitors will be the approach used here. The proposed investigations are novel and fundamental to the development of gene based therapy to inhibit myocyte apoptosis in the diabetic heart.